This invention is in the field of detection of molecular recognition events, in particular use of photopolymerization for amplification and detection of these events.
A variety of methods exist for detection of molecular recognition events. Detection of molecular recognition events such as DNA hybridization, antibody-antigen interactions, and protein-protein interactions becomes increasingly difficult as the number of recognition events to be detected decreases. Of particular interest are molecular recognition events between a target and a probe.
One approach to the problem is to increase the number of recognition events taking place. For example, polymerase chain reaction (PCR) increases the number of copies of DNA or RNA to be detected. Other molecular biology techniques which increase the number of copies of DNA or RNA to be detected include reverse transcription polymerase chain reaction (RT-PCR), strand displacement amplification, and Eberwine linear amplification.
Another approach is to amplify the signal due to each molecular recognition event. For example, DNA detection methods based on oligonucleotide-modified particles have been reported (U.S. Pat. Nos. 6,740,491, 6,777,186, 6,773,884, 6,767,702, 6,759,199, 6,750,016, 6,730,269, 6,720,411, 6,720,147, 6,709,825, 6,682,895, 6,673,548, 6,667,122, 6,645,721, 6,610,491, 6,582,921, 6,506,564, 6,495,324, 6,417,340 and 6,361,944 and Park, S.-J. et al, 2002, Science, 295,5559, 1503-1506). U.S. Pat. No. 6,602,669 relates to silver staining nanoparticles.
DNA detection methods based on branched DNA have also been reported (U.S. Pat. Nos. 5,681,702, 5,597,909, 5,580,731, 5,359,100, 5,124,246, 5,545,730, 5,594,117, 5,571,670, 5,594,118, 5,681,697, 5,591,584, 5,571,670, 5,624,802, 5,635,352, and 5,591,584. The branched DNA assay is a solution phase assay that involves a number of probe oligonucleotides that bind to multiple sites on the target viral RNA. Detection is possible because each hybridization event is accompanied by the binding of a fluorophore (Kern, D., Collins, M., Fultz, T., Detmer, J., Hamren, S., Peterkin, J., Sheridan, P., Urdea, M., White, R., Yeghiazarian, T., Todd, J. (1996) “An Enhanced-sensitivity Branched-DNA Assay for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma” Journal of Clinical Microbiology 34:3196-3203). The synthetic effort required for this assay is relatively large: multiple probes are designed for each RNA of interest, and the assay depends on the binding of these probes to multiple preamplifier and amplifier molecules that also must be designed and synthesized.
Dendrimer-based DNA detection methods have also been reported (U.S. Pat. Nos. 5,710,264, 5,175,270, 5,487,973, 5,484,904 and Stears, R. et al., 2000, Physiol. Genomics 3: 93-99). Dendrimers are complexes of partially double-stranded oligonucleotides, which form stable, spherical structures with a determined number of free ends. Specificity of the dendrimer detection is accomplished through specific binding of a capture oligonucleotide on a free arm of the dendrimer. Other arms of the dendrimer are labeled for detection. This method does not require enzymes and can produce amplification of 300-400.
Tyramide signal amplification is reported in U.S. Pat. Nos. 6,593,100 and 6,372,937.
Rolling circle amplification has been described in the scientific literature (Baner et al. (1998) Nuc. Acids Res. 26:5073-5078; Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88:189-193; Lizardi et al. (1998), Nat. Genet. 19:225-232; Zhang et al., Gene 211:277 (1998); and Daubendiek et al., Nature Biotech. 15:273 (1997)). Rolling circle amplification is capable of detecting as few as 150 molecules bound to a microarray (Nallur, G., Luo, C., Fang, L., Cooley, S., Dave, V., Lambert, J., Kukanskis, K., Kingsmore, S., Lasken, R., Schweitzer, B. (2001) “Signal Amplification by Rolling Circle Amplification on DNA Microarrays” Nucleic Acids Research 29:E1 18). The main drawback to RCA is the necessity of DNA polymerase.
Ligase chain reaction is reported in U.S. Pat. Nos. 5,185,243 and 5,573,907.
Cycling probe technology is reported in U.S. Pat. Nos. 5,011,769, 5,403,711, 5,660,988, and 4,876,187.
Microfabricated disposable DNA sensors based on enzymatic amplification electrochemical detection was reported by Xu et al. (Xu et al., 2001, Electoanalysis, 13(10), 882-887).
Surface initiated polymerization from surface confined initiators has been reported. Biesalski et al. report poly(methyl methacrylate) brushes grown in situ by free radical polymerization from an azo-initiator monolayer covalently bound to the surface (Biesalski, M. et al., (1999), J. Chem. Phys., 111 (15), 7029). Surface initiated polymerization for amplification of patterned self-assembled monolayers by surface-initiated ring opening polymerization (Husemann, M. et al., Agnewandte Chemie Int. Ed. (1999), 38(5) 647-649) and atom transfer radical polymerization (Shah, R. R. et al., (2000), Macromolecules, 33, 597-605) has been also reported.
DNA microarrays, or biochips, represent promising technology for accurate and relatively rapid pathogen identification (Wang, D., Coscoy, L., Zylberberg, M., Avila, P. C., Boushey, H. A., Ganem, D., DeRisi, J. L. (2002) “Microarray Based Detection and Genotyping of Viral Pathogens,” PNAS, 99(24), 15687-15692). Anthony et al. recently demonstrated rapid identification of 10 different bacteria in blood cultures using a BioChip (Anthony, R. M., Brown, T. J., French, G. L. (2000) “Rapid Diagnosis of Bacteremia by Universal Amplification of 23S Ribosomal DNA Followed by Hybridization to an Oligonucleotide Array” Journal of Clinical Microbiology 38:781-788). The microarray assay was conducted in ˜4 hrs. The approach utilized universal primers for PCR amplification of the variable region of bacterial 23s ribosomal DNA, and a 3×10 array of 30 unique capture sequences. This work demonstrates an important aspect of BioChip platforms—the capability to screen for multiple pathogens simultaneously. DeRisi and co-workers demonstrated a “virus chip” that contained sequences for hundreds of viruses, including many that cause respiratory illness (Wang et al., 2002). This chip proved useful in identifying the corona virus associated with SARS (Risberg, E. (2003) “Gene Chip Helps Identify Cause of Mystery Illness,” USA Today (Jun. 18, 2003)). Evans and co-workers have demonstrated that a DNA microarray could be used for typing and sub-typing human influenza A and B viruses (Li, J., Chen, S., & Evans, D. H. (2001) “Typing and Subtyping Influenza Virus Using DNA Microarrays and Multiplex Reverse Transcriptase PCR” Journal of Clinical Microbiology 39:696-704). In both the DeRisi and Evans work PCR technology was used to amplify the genetic material for capture and relatively expensive fluorescent labels (˜$50 in labels per chip) were used to generate signals from positive spots.
There remains a need in the art for relatively inexpensive labeling and signal amplification methods for molecular recognition events which do not require the use of enzymes for amplification. These methods would be useful in combination with DNA microarrays.